We have been intensively studying two regulatory elements of the human c-myc proto-oncogene and the proteins which bind to them. 1) A cell type and differentiation specific positive cis-element, FUSE, resides 1.5 kb upstream of promoter P1. The FUSE is single stranded when c-myc is expressed in vivo and this region is devoid of nucleosomes except when c-myc is repressed. A sequence specific, single strand DNA binding protein, FUSE binding protein, FBP, interacts with this element. Cloning of FBP revealed a protein comprised of alternating amphipathic helices (five) and repeating units (four). FBP possess the ability to displace oligonucleotides bearing its binding site from DNA, and hence can invade a double helix. Potent transcription activation and repression domains have been identified and analyzed. FBP can bind to duplex FUSE, when this element is supercoiled. Characterization of FBP2 and FBP3 have revealed that these siblings of FBP possess generally similar structures and properties. 2) A complex set of factors binds 100-150 bp upstream of P1 to a cytidine-rich nonanucleotide sequence, repeated five times. This region is required for transcription from P1, augments P2 mediated expression and stimulates reporter gene expression when fused to a heterologous promoter. Surprisingly, several sequence specific, single strand DNA binding proteins recognize this region. One of these proteins, binding to the pyrimidine-rich strand is hnRNP K. hnRNP K binds more tightly to ssDNA than to RNA. hnRNP K also interacts specifically with the CT-element in supercoiled DNA. In vitro transcription experiments and transfection studies indicate that hnRNP K participates in the stimulation of gene expression through the CT-element. hnRNP K interacts with molecules in the TFIID fraction, including TBP. hnRNP K also binds to the tumor suppressor protein, retinoblastoma--Rb. Binding to the opposite strand are protein products of alternately spliced mRNAs encoding a zinc finger protein. 3) The properties of FBP and hnRNP K have caused us to look at the state of c-myc regulatory cis-elements in vivo, demonstrating single strand features at several sites. Experiments analyzing the topological properties of several cis-elements are in progress. 4) Experiments are in progress to develop systems to test the coupling of the activity of particular cis-elements and their associated single strand or supercoil specific binding proteins in vivo.